This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During the past year my lab has focused on specific aim #1 and specific aim #3, experiment 2 of my grant. Specific aim #1 is to identify the regulatory elements driving neural crest-specific expression of the chicken homology of FoxD3 (cFoxd). To this end we cloned an approximately 3kb genomic DNA fragement 5'to cFoxD3, inserted it into the pBGZA reporter vector, and used in ovo electroporation to transfect the construct into the dorsal neural tube of stage 11 chicken embryos. This construct did not drive reporter gene expression the neural tube or neural crest cells, so this summer we will begin cloning larger 5'fragments and some 3'fragments to test. Specific aim #3, experiment 2 involves determining whether the regulatory elements from AmphifoxD are capable of driving reporter gene expression in the vertebrate neural tube and/or neural crest cells. Our data thus far indicate that they are not, suggesting that that the difference in expression of foxD3 between amphioxus and vertebrate embryos is due primarily to cis-regulatory factors rather than trans-regulatory factors. In addition to continuing our work on specific aim #3, we will begin work on specific aim #2 (generating constitutive activator and repressor forms of cFoxD3) and specific aim #3, experiment 1 (testing the ability of AmphifoxD to induce ectopic neural crest cells when expressed in the vertebrate neural tube). I will be doing this work with Rachel Patterson, a sophomore Biology major and my first INBRE scholar.